RESUMO
Peripheral tethering factors bind to small GTPases in order to obtain their correct location within the Golgi apparatus. Using fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) we visualized interactions between Arabidopsis homologues of tethering factors and small GTPases at the Golgi stacks in planta. Co-expression of the coiled-coil proteins AtGRIP and golgin candidate 5 (GC5) [TATA element modulatory factor (TMF)] and the putative post-Golgi tethering factor AtVPS52 fused to green fluorescent protein (GFP) with mRFP (monomeric red fluorescent protein) fusions to the small GTPases AtRab-H1(b), AtRab-H1(c) and AtARL1 resulted in reduced GFP lifetimes compared to the control proteins. Interestingly, we observed differences in GFP quenching between the different protein combinations as well as selective quenching of GFP-AtVPS52-labelled structures. The data presented here indicate that the FRET-FLIM technique should prove invaluable in assessing protein interactions in living plant cells at the organelle level.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fatores de Transcrição/metabolismo , Animais , Arabidopsis , Proteínas de Arabidopsis/genética , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Proteínas de Ligação a DNA/genética , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tiazolidinas/metabolismo , Nicotiana/anatomia & histologia , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Ypt/Rab GTPases act as key regulators of intracellular traffic through the conformational differences exhibited by their GTP or GDP-bound forms. In this paper, two Arabidopsis Ypt6 homologues, AtRAB-H1(b) and AtRAB-H1(c) were characterized and compared. Using a live cell imaging approach, it is shown that yellow fluorescent protein-fusions (YFP) of AtRAB-H1(b) and AtRAB-H1(c) locate to the Golgi and to the cytosol in both Nicotiana tabacum and in Arabidopsis thaliana. In addition, YFP-AtRAB-H1(b) targets an as yet unknown compartment not labelled by YFP-AtRAB-H1(c) or Golgi markers. It is also shown that the subcellular location of YFP-AtRAB-H1(b) and YFP-AtRAB-H1(c) is affected by the state of GTP-binding and that expression of a GTP-deficient mutant results in increased apoplastic fluorescence of a secretory form of YFP.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Expressão Gênica , Genes Reporter , Complexo de Golgi/metabolismo , Nicotiana/genética , Proteínas rab de Ligação ao GTP/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Citosol/metabolismo , Complexo de Golgi/genética , Guanosina Trifosfato/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Nicotiana/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
New findings reveal that many membrane proteins undergo regulated trafficking between intracellular compartments and the plasma membrane. This also appears to be a common regulatory mechanism in the control of cell wall metabolism.
